In vitro reconstruction of the mitochondrial translation system of yeast.

We have isolated the translation system from yeast mitochondria and have reconstructed it in vitro. This submitochondrial system, composed of mitochondrial ribosomes, tRNA, pH 5 fraction and mRNA, is maximally active at 10 mM Mg2+ and 100 mM KCl or NH4Cl. NH4+ is more stimulatory than K+. Added Escherichia coli tRNA gives less than half the activity obtained with added mitochondrial tRNA. Activity is enhanced with protease inhibitors but not with Ca2+, spermine, or spermidine. In contrast to heterologous translation systems, the present system produces products with molecular weights similar to those of products synthesized by yeast mitochondria in vivo and by intact yeast mitochondria in vitro. The results support the idea that the unique coding features of the mitochondrial genome require a unique translation system for accurate translation of mitochondrial mRNAs.